Our long-term objective is to develop effective gene therapies for the treatment of malignancy and melanoma in particular. We have focused both on the discovery of novel genes able to kill tumor cells locally with concomitant activation of specific immune effector cells and on the development of efficient and targeted vectors for their delivery to tumors in situ. The goal of this proposal is to build on work supported by NIH grant RO1 CA 85931. Therein, we successfully developed a novel, highly potent class of genes, the Fusogenic Membrane Glycoproteins (FMG), for gene/ immunotherapy of cancer. Vaccines of FMG-mediated fusing tumor cells significantly increased survival of tumor-bearing mice in both prophylactic and therapy models, leading us to develop a clinical trial at the Mayo Clinic in Stage IV melanoma patients. Consistent with these data, we also described a novel class of subcellular vesicles - syncytiosomes -released during non apoptotic syncytial killing which deliver antigens into dendritic cells for cross presentation to T cells and are effective immunogens in vivo. We also used FMG gene transfer to identify and isolate a completely novel class of small tumor cell/dendritic cell hybrids which cure well established metastatic disease in murine models. Finally, transcriptionally targeted plasmid and retroviral vectors were constructed which combine both local cytotoxic and highly immunostimulatory gene therapy for melanoma. Concurrently, we observed a significant enhancement of virotherapy using replication competent adenoviral vectors in the presence of fusion both in vitro and in vivo. We now propose to build on these data from grant RO1 CA85931. As summarized in Figure 16 of the current proposal, we will 1: Characterize the immunostimulatory components and functions of syncytiosomes; 2: Characterize the interactions of dendritic cells with fusing tumor cells at the level both of uptake of tumor antigen and/or through the formation of tumor cell/DC hybrids to ,generate improved cancer vaccines; 3: Generate both replication incompetent and conditionally replication competent adenoviral vectors with targeted intratumoral expression of FMG to improve both vector performance and therapeutic efficacy. Data from these Specific Aims will allow us to proceed directly to further Phase I/ll clinical trials, either in the context of cell free, syncytiosome-based vaccines, and/or dendritic cell/tumor fusing cell vaccines and/or direct intratumoral injection of melanoma-targeted, FMG expressing adenoviral vectors